Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

Abstract

Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra-to the intracellular milieu.

Role of Intracellular Carbon Metabolism Pathways in Shigella flexneri Virulence

“…In this study, we show that blocking either an early (Pfk) or late (Pyk) step in the EMP pathway resulted in loss of wild-type plaque formation. The importance of this glycolytic pathway for intracellular growth was supported by the observation that enzymes for the pathway were found at higher levels in intracellular S. flexneri than in bacteria grown in vitro (24), even though transcriptome analysis showed reduced expression of the genes (20). The EMP pathway may be important not only in using sugars as an energy source but also in the production of essential metabolic intermediates.…”

“…Extracellular bacteria microinjected into the eukaryotic cell cytoplasm fail to replicate (19), suggesting that intracellular cytosolic pathogens have adaptations for growth inside cells. This is supported by studies of the transcriptome and proteome of intracellular shigellae, which reveal specific responses to growth in the intracellular environment (20)(21)(22)(23)(24). For example, the increased expression of iron transport genes and high levels of iron transport proteins indicate that the eukaryotic cytosol is an iron-restricted environment.…”

“…There is reduced expression of genes encoding the glucose uptake proteins ptsG, manX, manY, and manZ during intracellular growth (22,23). However, analysis of the proteome of intracellular S. flexneri showed that two of these proteins (ManZ and ManX), as well as other proteins involved in sugar transport (FruA and FruB), were present at high levels in intracellular bacteria (24). The relative abundance of these proteins suggests that the bacteria can import sugars during intracellular growth.…”

“…Plaque assays were performed as previously described (24). Briefly, S. flexneri was grown as described for the invasion assay.…”

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

“…It is also noteworthy that studies of closely related bacterial pathogens (i.e., Escherichia coli or Shigella spp.) have expanded our knowledge on bacterial adaptations upon interactions with their host cells (20,21,22,23,24,25). For instance, Pieper et al studied the Shigella flexneri proteome within infected epithelial cells (24).…”